Journal: Cell communication and signaling : CCS
Article Title: Repression of Connexin26 hemichannel activity protects the barrier function of respiratory airway epithelial cells against LPS-induced alteration.
doi: 10.1186/s12964-025-02228-6
Figure Lengend Snippet: Fig. 2 Molecular docking of CVB4-57 in the structure of Cx26 hemichannels. a-b Putative binding modes of CVB4-57 on the Cx26 7QEV structure (clipped molecular surface) based on the distribution of best docked poses in site-01 (a) and site-02 (b). NTHs, relative to site-01 cavity (left NTH in brown) and site- 02 cavity (between NTHs) are shown in brown and gold (tubes, ribbons), respectively. Some tagged residues (navy blue) are shown below the molecular surface (transparency). For the molecular docking of CVB4-57 in site-01 (a), three pathways could be recorded: linear along the cavity (pink pose); con strained (green poses); extended towards site-02 cavity (purple). For the molecular docking in site-02 (b), a main linear fluctuation was recorded near the NTH (right, pink-purple). Poses were found in the central sub-cavity (teal) and in site-01 (light green). c-d Since the Cx26 structure 7QEV consists of only two chains (a, b, tan color), three 7QEV structures (tan, teal, blue) were aligned on 7QEW [35] (Cx26 type, hexamer) to reconstruct a hexamer. c Clipped hexamer (orthogonal, view from cytoplasmic side) showing best docked poses in site-01 (left, under brown ribbons) and site-02 (right, near gold ribbons). One linear pose of CVB4-57 per site was highlighted by molecular surface (site-01: purple, site-02: dark grey). d Laterally clipped hexamer showing the pore and molecular surfaces (transparency) of highlighted docked poses. The position of Ca2+ atoms (green spheres) is also shown after alignment with 5ER7 [54] (Cx26 type, dodecamer split as hexamer)
Article Snippet: The primary antibodies anti-Cx26 antibody (4 μg/mL, Alomone Labs; ACC-212), anti-Cx43 antibody (0.75 μg/mL, SigmaAldrich, C6219), anti-TLR4 antibody (0.5 μg/mL, Thermo Fisher Scientific, 48-2300), anti-CLDN1 (5 μg/mL, Thermo Fisher Scientific, 51-9000), anti-CLDN3 (3 μg/ mL, Thermo Fisher Scientific, 34-1700), anti-CLDN4 (2 μg/mL, Thermo Fisher Scientific, 32-9400) and antiZO-1 (1.25 μg/mL, Thermo Fisher Scientific, 40-2200) were diluted in PBS and added to the cells overnight at 4°C.
Techniques: Binding Assay
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![Fig. 1 LPS enhances the EtdBr dye uptake in airway epithelial cells. a-b EtdBr dye uptake rates (fluorescence intensities (AU)/min) under different perfu sion conditions (exemplary experiment see Fig. S1) in Calu-3 cells (a) and PBEPCs (b) cultivated under control conditions or with 1 ng/mL LPS for 24 h. Perfusion conditions: 2 mM [Ca2+]ex, 0 mM [Ca2+]ex or 0 mM [Ca2+]ex + 1 mM La3+ (n = cell patches for Calu-3 cells, single cells for PBPECs). Kruskal-Wallis test with Dunn’s multiple comparison test (p < 0.05 *, p < 0.001 *** vs. different perfusion conditions; p < 0.05 #, p < 0.001 ### vs. control). c Real-time qRT-PCR for different Cx isoforms in PBEPCs. d Immunofluorescence staining against <t>Cx26</t> or Cx43 (yellow) in PBEPCs. Scale bar = 20 μm. e Exemplary immunofluo rescence staining against Cx26 (yellow) in the airways of PCLS cultivated under control conditions or with 1 µg/mL LPS for 3 h. Scale bar = 10 μm. f Cx26 immunofluorescence signal (particle count/area, relative to control) in PCLS after treatment with 1 µg/mL LPS for 3 h (n = analyzed PCLS from 3 donors, 2 PCLS/donor with mean of 3 airway areas/PCLS). Different symbol shapes visualize different donors. Unpaired two-tailed Student’s t-test (p < 0.05 * vs. con trol). g EtdBr dye uptake rates in absence of [Ca2+]ex relative to the rates obtained in presence of [Ca2+]ex in Calu-3 cells in which Cx26 or Cx43 expression was suppressed using respective siRNA and which were treated with 1 ng/mL LPS or vehicle for 24 h (n = measured cell patches). Kruskal-Wallis test with Dunn’s multiple comparison test (p < 0.01 **, p < 0.001 *** vs. negative siRNA + vehicle; p < 0.05 #, p < 0.001 ### vs. respective siRNA + vehicle; p < 0.001 +++ vs. LPS). h EtdBr dye uptake rates in absence of [Ca2+]ex ± 5 µM CVB4-57 relative to the rates obtained in presence of [Ca2+]ex in Calu-3 cells treated with vehicle or 1 ng/mL LPS for 24 h (n = cell patches). Kruskal-Wallis test with Dunn’s multiple comparison test (p < 0.001 *** vs. vehicle; p < 0.001 ### vs. LPS)](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_0298/pm40380298/pm40380298__page5_image1.jpg)
![Fig. 2 Molecular docking of CVB4-57 in the structure of Cx26 hemichannels. a-b Putative binding modes of CVB4-57 on the Cx26 7QEV structure (clipped molecular surface) based on the distribution of best docked poses in site-01 (a) and site-02 (b). NTHs, relative to site-01 cavity (left NTH in brown) and site- 02 cavity (between NTHs) are shown in brown and gold (tubes, ribbons), respectively. Some tagged residues (navy blue) are shown below the molecular surface (transparency). For the molecular docking of CVB4-57 in site-01 (a), three pathways could be recorded: linear along the cavity (pink pose); con strained (green poses); extended towards site-02 cavity (purple). For the molecular docking in site-02 (b), a main linear fluctuation was recorded near the NTH (right, pink-purple). Poses were found in the central sub-cavity (teal) and in site-01 (light green). c-d Since the Cx26 structure 7QEV consists of only two chains (a, b, tan color), three 7QEV structures (tan, teal, blue) were aligned on 7QEW [35] (Cx26 type, hexamer) to reconstruct a hexamer. c Clipped hexamer (orthogonal, view from cytoplasmic side) showing best docked poses in site-01 (left, under brown ribbons) and site-02 (right, near gold ribbons). One linear pose of CVB4-57 per site was highlighted by molecular surface (site-01: purple, site-02: dark grey). d Laterally clipped hexamer showing the pore and molecular surfaces (transparency) of highlighted docked poses. The position of Ca2+ atoms (green spheres) is also shown after alignment with 5ER7 [54] (Cx26 type, dodecamer split as hexamer)](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_0298/pm40380298/pm40380298__page10_image1.jpg)
![Fig. 3 Involvement of TLR4 and TNF-α signaling in the LPS-induced enhancement of Cx26 hemichannel activity. a Real-time qRT-PCR experiments for TLR4 mRNA expression in Calu-3 cells and PBEPCs (n = 3 biological replicates or donors, respectively). b Immunofluorescence staining against TLR4 (yel low) in Calu-3 cells and PBEPCs. Scale bar = 20 μm. c EtdBr dye uptake rates in absence of [Ca2+]ex relative to the rates obtained in presence of [Ca2+]ex in Calu-3 cells treated for 24 h with vehicle, 1 ng/mL LPS ± preincubation (0.5 h) with 20 µM C34 or C34 alone (n = cell patches). Kruskal-Wallis test with Dunn’s multiple comparison test (p < 0.001 *** vs. vehicle, p < 0.001 ### vs. LPS). d Real-time qRT-PCR for TNF-α mRNA amount in Calu-3 cells treated for 3 h with 1 µg/mL LPS (n = 3). Unpaired two-tailed Student’s t-test. e EtdBr dye uptake rates in absence of [Ca2+]ex relative to the rates obtained in pres ence of [Ca2+]ex in Calu-3 cells treated for 24 h with vehicle, 1 ng/mL LPS ± preincubation (0.5 h) with 10 µM SPD-304 or 10 µM Marimastat, with SPD-304/ Marimastat alone or for 1 h with 10 ng/mL TNF-α (n = cell patches). Kruskal-Wallis test with Dunn’s multiple comparison test (p < 0.05 *, p < 0.001 *** vs. vehicle; p < 0.001 ### vs. LPS; p < 0.001 +++ vs. TNF-α). f EtdBr dye uptake rates in absence of [Ca2+]ex relative to the rates obtained in presence of [Ca2+]ex in Calu-3 cells in which Cx26 expression was suppressed using respective siRNA and which were treated with 10 ng/mL TNF-α or vehicle for 1 h (n = cell patches). Kruskal-Wallis test with Dunn’s multiple comparison test (p < 0.01 **, p < 0.001 *** vs. negative siRNA + vehicle; p < 0.05 #, p < 0.001 ### vs. respec tive siRNA + vehicle; p < 0.001 +++ vs. TNF-α)](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_0298/pm40380298/pm40380298__page11_image1.jpg)
